| Abstract: |
The PriA replication protein of Escherichia coli (formerly replication factor Y or protein n') is multifunctional. It is a site-specific, single- stranded DNA-dependent ATPase (dATPase), a 3' → 5' DNA helicase, and guides the ordered assembly of the primosome, a mobile, multiprotein DNA replication priming/helicase complex. Although PriA is not absolutely required for viability, priA null mutant cells grow very slowly, have poor viability, and form extensive filaments. In order to assess which of the multiple activities of PriA are required for normal replication and growth, site-directed mutagenesis was employed to introduce single amino acid substitutions for the invariant lysine within the consensus nucleotide-binding motif found in PriA. Biochemical characterization of the representative purified mutant PriA proteins revealed them to be completely deficient in nucleotide hydrolysis, incapable of translocation along a single-stranded DNA binding protein-coated single-stranded DNA template, and unable to manifest the 3' → 5' DNA helicase activity of wild-type PriA. These mutant proteins were, however, capable of catalyzing the assembly of active primosomes in vitro. Furthermore, when supplied in trans to insertionally inactivated priA cells, plasmids containing a copy of these mutant priA genes restored the wild-type growth rate, viability, and cell morphology. Based on these results, a model for PriA function in vivo is discussed. |
| Keywords: |
controlled study; dna binding protein; mutation; dna-binding proteins; nonhuman; dna replication; cell structure; amino acid substitution; enzyme activity; bacterial protein; molecular sequence data; escherichia coli; base sequence; plasmids; mutagenesis, site-directed; helicase; catalysis; dna binding; adenosine triphosphatase; site directed mutagenesis; hydrolysis; dna, bacterial; dna helicases; lysine; electrophoresis, polyacrylamide gel; bacterial growth; bacterium mutant; dna template; priority journal; article; support, u.s. gov't, p.h.s.; adenosinetriphosphatase
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